Regulation of intracellular calcium concentration is vitally important to a wide range of cell functions. This project is designed to measure rapid changes in intracellular pH and intracellular calcium in sperm of the sea urchin Strongylocentrotus purpuratus. Changes in these parameters accompany modifications of the sperm which ready it for fusion with the egg to initiate development of a new organism. I have applied optically detected fluorescent dyes to systematically measure intracellular pH (with carboxyfluoresceins) or intracellular calcium (with fura-2 or indo-1) to investigate: 1) physiological effects of peptide from the egg which causes chemotaxis of the sperm, and 2) effects of a complex egg coat which causes the sperm acrosome reaction. The acrosome reaction includes exocytosis of the sperm acrosomal granule and is essential for fertilization. 1) Our results show that the chemoattractant peptide speract causes a rapid increase in intracellular pH that allows for a transient rise in intracellular calcium. Inhibition of the increase in pH inhibits the calcium entry. The increase in calcium is important because sperm chemotaxis does not occur in the absence of external calcium. 2) The morphological changes of the sperm acrosome reaction also follow increases in intracellular pH and calcium. The increase in intracellular calcium is larger than that induced by the chemoattractant and is not transient. Inhibitors which block the acrosome reaction partially inhibit both pH and calcium increases. These studies form a basis for identification of biochemical components of the sperm plasma membrane regulating movements of calcium and hydrogen ions. Initial studies with monoclonal antibodies implicate a sperm plasma membrane protein of 210 kDa as important to the regulation of sperm calcium metabolism.